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Several robust and reliable procedures are available, relying on chemical modification inducing RT stops or nucleotide misincorporations during reverse transcription. Others are based on cleavage reactions and RT stop signals. However, these methods address only one side of the RT stop or misincorporation position. Here, we describe Led-Seq, a new approach based on lead-induced cleavage of unpaired RNA positions, where both resulting cleavage products are investigated. The RNA fragments carrying 2\u2032, 3\u2032-cyclic phosphate or 5\u2032-OH ends are selectively ligated to oligonucleotide adapters by specific RNA ligases. In a deep sequencing analysis, the cleavage sites are identified as ligation positions, avoiding possible false positive signals based on premature RT stops. With a benchmark set of transcripts in Escherichia coli, we show that Led-Seq is an improved and reliable approach based on metal ion-induced phosphodiester hydrolysis to investigate RNA structures in vivo.<\/jats:p>","DOI":"10.1093\/nar\/gkad312","type":"journal-article","created":{"date-parts":[[2023,4,28]],"date-time":"2023-04-28T13:35:33Z","timestamp":1682688933000},"page":"e63-e63","source":"Crossref","is-referenced-by-count":1,"title":["Led-Seq:\u00a0ligation-enhanced double-end sequence-based structure analysis of RNA"],"prefix":"10.1093","volume":"51","author":[{"ORCID":"http:\/\/orcid.org\/0000-0001-9828-8329","authenticated-orcid":false,"given":"Tim","family":"Kolberg","sequence":"first","affiliation":[{"name":"Institute for Biochemistry, Leipzig University , Br\u00fcderstr. 34, 04103 Leipzig, Germany"}]},{"given":"Sarah","family":"von\u00a0L\u00f6hneysen","sequence":"additional","affiliation":[{"name":"Bioinformatics Group, Department of Computer Science and Interdisciplinary Center for Bioinformatics, Leipzig University , H\u00e4rtelstr. 16\u201318, 04107 Leipzig, Germany"}]},{"given":"Iuliia","family":"Ozerova","sequence":"additional","affiliation":[{"name":"Bioinformatics Group, Department of Computer Science and Interdisciplinary Center for Bioinformatics, Leipzig University , H\u00e4rtelstr. 16\u201318, 04107 Leipzig, Germany"}]},{"given":"Karolin","family":"Wellner","sequence":"additional","affiliation":[{"name":"Institute for Biochemistry, Leipzig University , Br\u00fcderstr. 34, 04103 Leipzig, Germany"}]},{"given":"Roland\u00a0K","family":"Hartmann","sequence":"additional","affiliation":[{"name":"Institute for Pharmaceutical Chemistry, Philipps University Marburg , Marbacher Weg 6,\u00a035037 Marburg, Germany"}]},{"ORCID":"http:\/\/orcid.org\/0000-0002-5016-5191","authenticated-orcid":false,"given":"Peter\u00a0F","family":"Stadler","sequence":"additional","affiliation":[{"name":"Bioinformatics Group, Department of Computer Science and Interdisciplinary Center for Bioinformatics, Leipzig University , H\u00e4rtelstr. 16\u201318, 04107 Leipzig, Germany"},{"name":"Max Planck Institute for Mathematics in the Sciences , Inselstra\u00dfe 22, D-04103 Leipzig, Germany"},{"name":"Department of Theoretical Chemistry, University of Vienna , W\u00e4hringerstra\u00dfe 17, A-1090 Wien, Austria"},{"name":"Facultad de Ciencias, Universidad Nacional de Colombia , Bogot\u00e1, Colombia"},{"name":"Santa Fe Institute , 1399 Hyde Park Rd., Santa Fe, NM\u00a087501, USA"}]},{"ORCID":"http:\/\/orcid.org\/0000-0003-0972-9386","authenticated-orcid":false,"given":"Mario","family":"M\u00f6rl","sequence":"additional","affiliation":[{"name":"Institute for Biochemistry, Leipzig University , Br\u00fcderstr. 34, 04103 Leipzig, Germany"}]}],"member":"286","published-online":{"date-parts":[[2023,4,28]]},"reference":[{"key":"2023062304133234100_B1","doi-asserted-by":"crossref","first-page":"849","DOI":"10.1016\/0092-8674(83)90117-4","article-title":"The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme","volume":"35","author":"Guerrier-Takada","year":"1983","journal-title":"Cell"},{"key":"2023062304133234100_B2","doi-asserted-by":"crossref","first-page":"1169","DOI":"10.1080\/15476286.2015.1094599","article-title":"New hypotheses derived from the structure of a flaviviral Xrn1-resistant RNA: conservation, folding, and host adaptation","volume":"12","author":"Kieft","year":"2015","journal-title":"RNA Biol."},{"key":"2023062304133234100_B3","doi-asserted-by":"crossref","first-page":"776","DOI":"10.1038\/nrg2172","article-title":"Ribozymes, riboswitches and beyond: regulation of gene expression without proteins","volume":"8","author":"Serganov","year":"2007","journal-title":"Nat. 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